HMGB1–C1q complexes regulate function by switching between leukotriene and specialized proresolving mediator biosynthesis

Tianye Liua,b, Alec Xianga, Travis Penga, Amanda C. Doranc,d,e, Kevin J. Traceyf, Betsy J. Barnesa, Ira Tabasc,d,e, Myoungsun Sona,b,1,2, and Betty Diamonda,b,1,2

aCenter for Autoimmune Musculoskeletal and Hematopoietic Diseases, The Feinstein Institute for Medical Research, Manhasset, NY 11030; bMD/PhD Program at Donald and Barbara Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY 11549; cDepartment of Medicine, Columbia University, New York, NY 10032; dDepartment of Physiology, Columbia University, New York, NY 10032; eDepartment of Pathology and Cell Biology, Columbia University, New York, NY 10032; and fThe Center for Biomedical Science, The Feinstein Institute for Medical Research, Manhasset, NY 11030

Edited by Lawrence Steinman, Stanford University School of Medicine, Stanford, CA, and approved September 9, 2019 (received for review May 2, 2019) Macrophage polarization is critical to and resolution contrast, when 5-LO is not phosphorylated, it localizes in the of inflammation. We previously showed that high-mobility group cytoplasm and promotes SPM production (15). Resolvins, es- box 1 (HMGB1) can engage for advanced glycation end pecially resolvin D1 (RvD1) (7S,8R,17S-trihydroxy-docosa- product (RAGE) to direct to a proinflammatory phenotype 4Z,9E,11E,13Z,15E,19Z-hexaenoic acid), contribute to resolu- characterized by production of type 1 IFN and proinflammatory tion of inflammation by promoting nuclear exclusion of 5-LO . In contrast, HMGB1 plus C1q form a tetramolecular and, in a positive-feedback loop, by enhancing production of complex cross-linking RAGE and LAIR-1 and directing monocytes theseSPMs.OtherSPMssuchasprotectinsormaresinsdonot to an antiinflammatory phenotype. Lipid mediators, as well as directly require 5-LO activation (15, 16). The antiinflammatory cytokines, help establish a milieu favoring either inflammation effects of lipoxin A4 (LXA4)(5S,6R,15S-trihydroxy-eicosa- or resolution of inflammation. This study focuses on the induction 7E,9E,11Z,13E-tetraenoic acid), RvD1, RvD2 (7S,16R,17S- of lipid mediators by HMGB1 and HMGB1 plus C1q and their trihydroxy-docosa-4Z,8E,10Z,12E,14E,19Z-hexaenoic acid), and regulation of IRF5, a critical for the induction INFLAMMATION their ability to reduce production of inflammatory cytokines IMMUNOLOGY AND and maintenance of proinflammatory . Here, we show such as IFN-γ,TNFα,andIL-1β are well established (17–19). that HMGB1 induces leukotriene production through a RAGE- C1q is a modulator of inflammation and repair that can main- dependent pathway, while HMGB1 plus C1q induces specialized tain quiescence or cooperate with damage-associated proresolving lipid mediators lipoxin A4, resolvin D1, and resolvin molecular patterns (DAMPs) to induce antiinflammatory macro- D2 through a RAGE- and LAIR-1–dependent pathway. Leukotriene phages (20, 21). C1q is composed of globular heads and a collagen- exposure contributes to induction of IRF5 in a positive-feedback like tail that binds to the leukocyte-associated Ig-like receptor-1 loop. In contrast, resolvins (at 20 nM) block IRF5 induction and pre- (LAIR-1), a transmembrane of the Ig superfamily (22–24). vent the differentiation of inflammatory macrophages. Finally, we High-mobility group box 1 (HMGB1) is a DAMP that is elevated have generated a molecular mimic of HMGB1 plus C1q, which cross- – links RAGE and LAIR-1 and polarizes monocytes to an antiinflamma- in the serum of SLE patients (25 28). Extracellular HMGB1 binds tory phenotype. These findings may provide a mechanism to control nucleic acid cargo, engages the receptor for advanced glycation nonresolving inflammation in many pathologic conditions. Significance leukotriene | SPMs | IRF5 | HMGB1 | C1q Monocyte to macrophage differentiation is critical to inflamma- roinflammatory macrophages contribute to immune protec- tion and resolution of inflammation and can be regulated by Ption in infection, but also to disease pathogenesis in auto- high-mobility group box 1 (HMGB1) and HMGB1 plus C1q, re- immune diseases, , Alzheimer’sdisease,andmany spectively. These are 2 evolutionarily old and highly conserved conditions of chronic inflammation (1–4). Antiinflammatory molecules. While HMGB1 causes a positive-feedback loop be- macrophages are important for cessation of inflammation and tween the proinflammatory lipid mediator leukotriene B4 (LTB4) tissue repair (5). The degree and timing of proinflammatory and and IRF5, an important regulator of inflammatory macrophage antiinflammatory macrophage induction are critical for maintain- polarization, HMGB1 plus C1q increases production of specialized ing immune homeostasis (6, 7). There is, therefore, a need to proresolving lipid mediators (SPMs) lipoxin A4, resolvin D1, and understand how to harness relevant pathways in order to regulate resolvin D2, which prevent IRF5 transcription. As nonresolving inflammation is a common condition, we designed an HMGB1– resolution of inflammation. However, the underlying mechanisms C1q mimetic peptide that can polarize monocytes to an antiin- for macrophage polarization are incompletely characterized. flammatory phenotype in vitro and in vivo. During inflammation, leukotrienes, produced from arachidonic

acid by 5-lipoxygenase (5-LO), help regulate leukocyte trafficking, Author contributions: T.L., I.T., M.S., and B.D. designed research; A.X., T.P., and A.C.D. chemotaxis, and diapedesis from the bloodstream into injured performed research; T.L., I.T., M.S., and B.D. analyzed data; and T.L., K.J.T., B.J.B., I.T., M.S., tissue (8, 9). Specialized proresolving lipid mediators (SPMs), and B.D. wrote the paper. which include lipoxins, resolvins, protectins, and maresins, are also The authors declare no competing interest. produced from polyunsaturated fatty acid precursors (6, 7, 10, 11). This article is a PNAS Direct Submission. These molecules block migration and stimulate Published under the PNAS license. macrophage uptake of cellular debris, which are processes re- 1M.S. and B.D. contributed equally to this work. quired for the resolution of inflammation (12). The generation of 2To whom correspondence may be addressed. Email: [email protected] or bdiamond@ leukotriene B4 (LTB4)(5S,6Z,8E,10E,12R,14Z-5,12-dihydroxy- northwell.edu. 6,8,10,14-icosatetraenoic acid) requires the function of 5-LO. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. When 5-LO is phosphorylated, it translocates to the nuclear 1073/pnas.1907490116/-/DCSupplemental. membrane and promotes leukotriene production (13, 14). In

www.pnas.org/cgi/doi/10.1073/pnas.1907490116 PNAS Latest Articles | 1of10 Downloaded by guest on September 27, 2021 end product (RAGE), and transports nucleic acid to endosomal stimulated with HMGB1 plus C1q secreted significantly less LTB4 Toll-like receptors (TLRs) 7 and 9 (27, 29). It is known that than monocytes stimulated with HMGB1 alone. They instead HMGB1-induced macrophage polarization of monocytes de- produced SPMs such as LXA4, RvD2, and RvD1 (Fig. 1 B–D). pends on its interaction with 5-LO and LTB4 receptor BLT1 C1q alone did not lead to the induction of SPMs. Levels of SPMs pathways (30). were increased at 1 to 3 h and were maintained for up to 6 h. The transcription factor IFN regulatory factor 5 (IRF5) has been identified as a regulator of proinflammatory macrophage HMGB1 and C1q Reciprocally Regulate Phosphorylation and Nuclear polarization (31–33). IRF5 directly induces transcription of Localization of 5-LO. LTB4 is generated from arachidonic acid by proinflammatory cytokines and type I IFN while repressing nuclear 5-LO, and lipoxins are produced from arachidonic acid by transcription of IL-10 and TGFβ in macrophages (32, 34). cytosolic 5-LO (15). We, therefore, evaluated 5-LO localization in Polymorphisms of the IRF5 leading to high expression in HMGB1 and HMGB1-plus-C1q–treated monocytes and de- monocytes are associated with rheumatoid and sys- termined the ratio of nuclear to cytosolic 5-LO. 5-LO was localized temic erythematosus (SLE) (35). The relationship be- in perinuclear regions in HMGB1-exposed cells while it was lo- tween IRF5 expression and leukotriene or resolvin production calized in the cytosol of HMGB1-plus-C1q–exposed cells (Fig. 2A). has not been fully explored. Here, we further explore this re- Additionally, serine 271 phosphorylation of 5-LO, which promotes lationship. We also address the involvement of C1q and other its nuclear localization, was induced at 1 h by HMGB1 but not by SPMs in leukotriene production and macrophage polarization. HMGB1 plus C1q (Fig. 2B). This suggests that HMGB1 plus C1q prevents HMGB1-induced LTB4 synthesis by inhibiting phos- Results phorylation and nuclear localization of 5-LO.

HMGB1 Leads to LTB4 Production and HMGB1 Plus C1q Leads to SPM In order to understand whether the RAGE and LAIR-1 Production in Human Monocytes. We previously showed that pathways contribute to HMGB1 induction of LTB4, we trans- HMGB1 induces the production of proinflammatory cytokines by fected human monocytes with RAGE-specific or LAIR-1–specific human monocytes in a RAGE-dependent pathway (20). We, small interfering RNA (siRNA). LTB4 production induced by therefore, asked whether leukotriene production is also a com- HMGB1 was significantly decreased in monocytes transfected ponent of the proinflammatory phenotype of HMGB1-stimulated RAGE-siRNA compared to control siRNA-transfected monocytes – monocytes. We assessed LTB4 in culture supernatant of HMGB1- but not in LAIR-1 knockdown monocytes, confirming a requirement A stimulated monocytes. LTB4 production was significantly induced of RAGE in HMGB1-stimulated LTB4 production (Fig. 3 ). by HMGB1 at 1 h. While it decreased at 3 h, it was still signifi- RvD2 production upon HMGB1-plus-C1q stimulation was di- cantly higher than in supernatant of unstimulated cells (Fig. 1A). minished in both RAGE-knockdown monocytes and in LAIR-1– Just as HMGB1 plus C1q suppresses induction of proinflammatory knockdown monocytes, respectively, showing that both RAGE cytokines and induces, instead, the production of IL-10, monocytes and LAIR-1 are required for HMGB1-plus-C1q–induced RvD2

Fig. 1. HMGB1 induces LTB4, and HMGB1 plus C1q induces SPMs in human monocytes. Induction of (A)LTB4,(B)LXA4,(C) RvD2, and (D) RvD1 by HMGB1 and/or C1q. Human monocytes were treated with HMGB1 (1 μg/mL) and/or C1q (25 μg/mL)inX-Vivo15serum-freemedium.LTB4,LXA4,RvD2(after1,3,and6h),and RvD1 (after 3 h) in supernatant were measured by ELISA (mean ± SD of triplicates; n = 3for1and6h;n = 5 for 3 h). ***P < 0.001; ns, not significant (1-way ANOVA).

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1907490116 Liu et al. Downloaded by guest on September 27, 2021 HMGB1-induced LTB4 secretion (Fig. 3H). RvD2 production in HMGB1-plus-C1q–triggered cells was not affected by IRF5 siRNA (SI Appendix,Fig.S1B). We then confirmed IRF5- dependent LTB4 secretion in mouse monocytes deficient in IRF5. HMGB1-induced LTB4 production was significantly less in IRF5-deficient monocytes than in WT monocytes (Fig. 3I). Reciprocally, when IRF5 was overexpressed in human monocytes, the basal production of LTB4 was increased, suggesting a role for IRF5 upstream of LTB4 (SI Appendix, Fig. S1C). These results suggest that the IRF5 pathway is the critical mediator of LTB4 production.

The Leukotriene Pathway Modulates IRF5 Transcription. To determine whether HMGB1-induced IRF5 expression is mediated by path- ways downstream of LTB4, we used U75302, an LTB4 receptor BLT1 inhibitor and LY255283, an LTB4 receptor BLT2 inhibi- tor to block leukotriene signaling (30, 37, 38). Blockade of BLT1 and BLT2, both individually and together led to a decrease in HMGB1-induced IRF5 transcription with the combination of in- hibitors having the strongest effect (Fig. 4A). To confirm the ob- servation, we showed that HMGB1-induced IRF5 transcription was abolished when either or both receptors were diminished by siRNA (Fig. 4B). We next asked whether LTB4 can induce IRF5 transcription. IRF5 transcription increased under LTB4 stimulation. BLT1 and BLT2 inhibitors, both individually and to- gether, decreased LTB -induced IRF5 transcription. (Fig. 4C). Fig. 2. HMGB1 and C1q reciprocally regulate phosphorylation and nuclear 4 Reciprocally, RvD1 and RvD2 (20 nM) and LXA4 (20 nM) localization of 5-LO. (A) Nuclear localization of 5-LO induced by HMGB1 and/or D C1q stimulation for 1 h; nucleus marked by PI (blue) and 5-LO stained with suppressed induction of IRF5 by HMGB1 (Fig. 4 ). Thus, SPMs INFLAMMATION IMMUNOLOGY AND rabbit anti–5-LO and Alexa Fluor 488 anti-rabbit IgG (red). The ratio of nuclear/ produced by 5-LO can prevent expression of IRF5 induced nonnuclear 5-LO was determined in more than 80 cells in each condition by HMGB1 stimulation. (mean ± SEM; n = 4). (Scale bar, 10 μm.) *P < 0.05 and **P < 0.01 (1-way ANOVA). (B) Immunoblot for serine-271 phosphorylation of 5-LO in total cell HMGB1-Induced Peritonitis. Having established pathways of lysates from HMGB1 or HMGB1-plus-C1q–stimulated monocytes (1 h). The fold leukotriene and SPM production in primary human monocytes, we induction of phospho-5-LO in HMGB1 and/or C1q-stimulated monocytes is next sought to explore mechanisms underlying proinflammatory compared to untreated samples from 3 independent experiments. *P < < and antiinflammatory macrophage polarization and the contribution 0.05 and **P 0.01 (1-way ANOVA). of lipid mediators in an in vivo mouse model. We determined that HMGB1, injected intraperitoneally (i.p.) into C57BL/6 mice, could μ production (Fig. 3A). We confirmed a requirement for RAGE in induce an inflammatory peritonitis. When HMGB1 (10 g/mouse) was injected i.p. into mice, the peritoneal exudate was character- LTB4 production (Fig. 3B) and a requirement for RAGE and C ized initially by increased levels of LTB4 followed by increasing LAIR-1 in RvD2 production in murine monocytes (Fig. 3 ). A SH2 domain-containing protein tyrosine phosphatase-1 (SHP-1) RvD2. (Fig. 5 ). In contrast, when mice were given HMGB1 is a tyrosine phosphatase that binds phosphorylated LAIR-1 (36). plus C1q i.p., there was more RvD2 in the peritoneal exudate andareductioninLTB.Wealsoobservedthatthelevelof To understand whether SHP-1 is critical to the production of 4 TNFα was high in HMGB1-induced exudates, while IL-10 was proresolving lipid mediators, we used siRNA to diminish SHP- high in HMGB1-plus-C1q–induced exudates (SI Appendix,Fig. 1 expression. HMGB1-plus-C1q–induced production of RvD2 in S2). These in vivo results are consistent with the data obtained human monocytes was decreased in SHP-1 siRNA-transfected D with human monocytes in which proinflammatory macrophages monocytes (Fig. 3 ). making leukotriene are generated by exposure to HMGB1 and antiinflammatory macrophages making SPMs by exposure to IRF5 Is Increased in HMGB1-Stimulated Macrophages and Positively HMGB1 plus C1q. Six hours after HMGB1 or HMGB1-plus-C1q Regulates LTB Production. To determine whether HMGB1- + − 4 injection, macrophages (CD11b Ly6G ) were isolated from peri- stimulated macrophages exhibit high expression of IRF5, as has toneal exudates. Macrophages from mice injected with HMGB1 been shown in proinflammatory macrophages triggered by en- exhibited a high level of TNFα and IRF5 mRNA, whereas mac- gagement of other surface receptors, we assessed the mRNA and rophages from mice injected with HMGB1 plus C1q exhibited a protein levels of IRF5 4 h after HMGB1 stimulation. IRF5 mRNA higher level of Mer, Arg1, and TGFβ mRNA, which are anti- and protein expression were increased in HMGB1-stimulated inflammatory markers in murine macrophages (Fig. 5B). Moreover, E macrophages (Fig. 3 ). In contrast, HMGB1 plus C1q did not a higher frequency of Merhigh macrophages was observed in mice induce IRF5. HMGB1-mediated IRF5 expression did not occur in injected with HMGB1 plus C1q than in mice injected with human monocytes transfected with RAGE siRNA, demonstrating HMGB1 (Fig. 5C). Merhigh and Merlow macrophages were isolated that IRF5 expression is downstream of RAGE engagement (Fig. and cultured ex vivo to confirm their proinflammatory or anti- F α low 3 ). TNF up-regulation was also blocked in monocytes trans- inflammatory phenotype. Mer cells produced more LTB4 than fected with RAGE siRNA (SI Appendix,Fig.S1A), confirming Merhigh cells, whereas Merhigh cells secreted more RvD2 than α low results of our previous studies, which showed that TNF induction Mer cells (Fig. 5D). We also examined the effect of LTB4 re- + − by HMGB1 was mediated through RAGE (33). We confirmed in ceptor antagonism in this peritonitis model. CD11b Ly6G cells RAGE-deficient and wild-type (WT) mouse monocytes that from peritoneal exudates of mice pretreated with LTB4 receptor RAGE is necessary for induction of IRF5 by HMGB1 (Fig. 3G). antagonists exhibited decreased TNFα and IRF5 mRNA and in- We next examined whether IRF5 modulates LTB4 production creased Mer and Arg1 mRNA compared to untreated controls in monocytes. We observed that IRF5-specific siRNA abrogated upon HMGB1 stimulation (Fig. 5E).

Liu et al. PNAS Latest Articles | 3of10 Downloaded by guest on September 27, 2021 Fig. 3. HMGB1 induces LTB4 through RAGE and IRF5, while HMGB1 plus C1q induces RvD2 through RAGE and LAIR-1. (A) LTB4 production by HMGB1 stimulation and RvD2 production by HMGB1-plus-C1q stimulation in RAGE or LAIR-1 siRNA-transfected human monocytes. Twenty-four hours after

transfection with siRNA, cells were stimulated with HMGB1 alone (1 μg/mL), C1q alone (25 μg/mL), or HMGB1 plus C1q. HMGB1-induced LTB4 (after 1 h) and HMGB1-C1q–induced RvD2 (after 3 h) were measured in culture supernatant by ELISA. Knockdown efficiencies of RAGE and LAIR-1 were assessed by qRT-PCR.

RE, relative expression. *P < 0.05 and ***P < 0.001 (1-way ANOVA or t test). n = 4. (B and C)LTB4 production by HMGB1 in RAGE-deficient monocytes and RvD2 production by HMGB1 plus C1q in RAGE- or LAIR-1–deficient monocytes. Splenic monocytes from WT mice or RAGE-deficient monocytes were stim-

ulated with HMGB1 for 1 h or HMGB1 plus C1q for 3 h. LTB4 and RvD2 measured by ELISA. *P < 0.05, **P < 0.01, and ***P < 0.001 (1-way ANOVA or t test). (D) Reduced RvD2 production by HMGB1-plus-C1q stimulation in SHP-1 siRNA-transfected human monocytes. Knockdown efficiency of SHP-1 were determined by qRT-PCR. n = 3. (E) Total cell lysates were subjected to Western blot with specific for IRF5 and β-actin. Numbers indicate the relative signal intensity compared to actin. Bar graphs show fold change compared to the intensity of IRF5 in untreated samples from 3 independent experiments. (F) RAGE-specific siRNA-transfected human monocytes showed significantly decreased IRF5 levels following HMGB1 stimulation for 4 h. IRF5 relative expression was de- termined by qRT-PCR. n = 4. (G) Splenic monocytes from WT mice or RAGE-deficient mice were isolated and stimulated with HMGB1 and/or C1q for 4 h. Each

dot represents an individual animal. (H) IRF5-specific siRNA abrogates HMGB1-stimulated LTB4 production 1 h after HMGB1 stimulation, n = 4. Knockdown efficiency of IRF5 was determined by qRT-PCR. (I) IRF5-deficient monocytes showed reduced LTB4 secretion following HMGB1 stimulation. Splenic monocytes from WT mice or IRF5-deficient mice were isolated and stimulated with HMGB1 and/or C1q for 1 h. Each dot represents an individual animal (mean ± SEM of 5 mice/group). *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant (1-way ANOVA).

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Fig. 4. LTB4 and RvD2 pathways reciprocally regulate IRF5 . (A)BlockadeoftheLTB4 pathway diminished HMGB1-induced IRF5 transcription. Human monocytes were preincubated with LTB4 receptor BLT1 antagonist (LY255283; 200 nM), LTB4 receptor BLT2 antagonist (U75304; 200 nM), or both for 15 min followed by HMGB1 for 4 h at 37 °C. IRF5 mRNA was assessed by qRT-PCR. n = 3. (B) Human monocytes transfected with siRNA for BLT1, BLT2, or both were treated with HMGB1 after 24 h for 4 h. Relative expression of IRF5 and knockdown efficiencies were determined by qRT-PCR. n = 3. (C) Human monocytes were preincubated with

LTB4 receptor BLT1 antagonist (LY255283; 200 nM) and/or LTB4 receptor BLT2 antagonist (U75304; 200 nM) for 15 min followed by LTB4 (200 nM) for 4 h. IRF5 mRNA assessed by qRT-PCR. (D)RvD1,RvD2,LXA4,andLXA4 analog suppress IRF5 mRNA. Human monocytes were preincubated with RvD1 (20 nM), RvD2 (20 nM), or LXA4 (20 nM) for 15 min followed by HMGB1 for 4 h. IRF5 mRNA induced by HMGB1 was assessed by qRT-PCR. n = 3. *P < 0.05; **P < 0.01; ***P < 0.001 (1-way ANOVA).

An HMGB1-Plus-C1q Mimetic Cross-Links RAGE and LAIR-1 and Induces incubated with HMGB1 plus RLCP, similar to HMGB1 plus C1q, Antiinflammatory Phenotypes. Having shown that HMGB1 induces exhibited decreased proinflammatory polarization and decreased LTB4 in monocytes in a RAGE-dependent pathway, and that this IRF5 mRNA compared to monocytes incubated with HMGB1 process, in turn, augments IRF5 transcription and a proinflammatory alone (Fig. 6D). RLCP in the presence of HMGB1 and by itself polarization of macrophages, and that HMGB1 plus C1q induces E – induced IL-10 transcription in human monocytes in vitro (Fig. 6 ). RvD2 in a RAGE- and LAIR-1 dependent pathway leading to WT mice injected with HMGB1 plus RLCP together produced suppression of IRF5 transcription and a proresolving polarization less LTB , less prostaglandin E (PGE ), and more RvD2 in their of macrophages both in vitro and in vivo, we wanted to develop a 4 2 2 molecule that could harness these pathways therapeutically. We, peritoneal exudates than those injected with HMGB1 alone after 3 h. (Fig. 6 F–H). Mice injected with RLCP had more alternatively therefore, generated a peptide containing a RAGE-binding region + + − of HMGB1 (B-box) (39), a short linker sequence (40), and a C1q activated macrophages (CD206 CD11b Ly6G ) in the peritoneal peptide that binds LAIR-1 and induces its phosphorylation (Fig. 6 exudatethanmiceinjectedwithHMGB1(Fig.6I). Thus, RLCP A and B). This peptide was able to cross-link RAGE and LAIR-1 functions similarly to HMGB1 plus C1q in both human monocytes in a proximity ligation assay (Fig. 6C) and hence is termed and in the mouse peritonitis model to induce antiinflammatory RAGE–LAIR-1 cross-linking peptide (RLCP). Human monocytes macrophage polarization.

Liu et al. PNAS Latest Articles | 5of10 Downloaded by guest on September 27, 2021 Fig. 5. C1q promotes antiinflammatory macrophages in HMGB1-induced peritonitis. C57BL/6 WT mice were given HMGB1 i.p. (10 μg/mouse) with or without C1q

(200 μg/mouse). (A) Peritoneal exudates from mice given HMGB1 and/or C1q were obtained at 3, 6, and 12 h, and LTB4 and RvD2 were measured by ELISA. Each dot + − represents an individual animal. (B) Six hours after injection with HMGB1 or HMGB1 plus C1q, CD11b Ly6G cells were isolated from peritoneal exudates and analyzed for TNFα, IRF5, Mer, Arg1, and TGFβ mRNA. n = 3. *P < 0.05, **P < 0.01, and ***P < 0.001 (t test). (C) The percentage of Mer-expressing cells in + − CD11b Ly6G cells was higher in mice injected with HMGB1 plus C1q than in mice injected with HMGB1 alone. n = 3. (D)IsolatedMerhigh and Merlow cells were

separately cultured in X-Vivo medium. LTB4 (1 h) and RvD2 (3 h) were measured in the culture supernatant. n = 3. (E)WTmicewereinjectedwithHMGB1i.p. (10 μg/mouse) or HMGB1-plus-BLT1 inhibitor (LY255283; 10 mg/kg). Peritoneal exudate cells were isolated, and proinflammatory and antiinflammatory markers were assessed on CD11b+Ly6G− cells by qRT-PCR (3 h). n = 3. *P < 0.05 (t test).

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Fig. 6. RAGE and LAIR-1 cross-linking peptide, RLCP, induces RvD2 and abolishes LTB4 induction by HMGB1. (A) Amino acid sequence of a fusion protein containing B box, a RAGE binding region of HMGB1 and a C1q tail peptide linked by a flexible (Gly4Ser)3 linker (RAGE and LAIR-1 cross-linking peptide [RLCP]). (B) RLCP phosphorylates LAIR-1 in a phospho-immunoreceptor array. RLCP (500 nM) was incubated with fresh human monocytes for 15 min at 37 °C. Relative quantification for the phosphorylation of LAIR-1 was normalized to control spots. n = 3. (C) Proximity ligation assay performed on human monocytes showed that RLCP (500 nM, 15 min) cross-links RAGE and LAIR-1. Red fluorescent dots represent proximity between RAGE and LAIR-1; blue represents nuclear stainingwith DAPI. Original magnification≥ 40×. One of 3 representative experiments is shown. (Scale bar, 10 μm.) (D and E) Human monocytes were incubated for 4 h with HMGB1 (1 μg/mL) and/or C1q (25 μg/mL), HMGB1 plus RLCP (500 nM), or RLCP alone (500 nM). IRF5 and IL-10 mRNA expression were assessed by qRT-PCR. n = 3. (F–H) C57BL/6 WT mice were given HMGB1 i.p. (10 μg/mouse) and/or C1q (200 μg/mouse), HMGB1 plus RLCP (500 μg/mouse), or RLCP (500 μg/mouse). Peritoneal

exudates were isolated, and LTB4 and PGE2 were measured by ELISA 3 h after injection. Each dot represents an individual animal (mean ± SEM). RvD2 was + + − measured by ELISA 6 h after injection. (I) Percentage of CD206 cells in CD11b Ly6G mouse peritoneal exudates from HMGB1 i.p. (10 μg/mouse), HMGB1 plus C1q (200 μg/mouse), or HMGB1 plus RLCP (500 μg/mouse). Each dot represents an individual animal. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant (1-way ANOVA).

Liu et al. PNAS Latest Articles | 7of10 Downloaded by guest on September 27, 2021 Together, these observations describe a positive-feedback loop 5-LO cellular localization and phosphorylation serves to explain in inflammatory macrophages (IRF5 and LTB4)andaninhibitory their observed effects on LTB4 production. We demonstrated pathway initiated by C1q (Fig. 7), suggesting that a balanced level that HMGB1 stimulation of monocytes can lead to an increase of HMGB1 and C1q is critical in immune homeostasis. in LTB4 production in a RAGE-dependent manner. It is known that RAGE-mediated vascular smooth muscle cell proliferation is Discussion enhanced by HMGB1 through 5-LO (47). Perinuclear localization The mammalian immune system relies on a fine balance of many of 5-LO is involved not only in leukotriene production but also in interrelated stimulatory and inhibitory pathways. Chronic in- the activation of NF-κB in granulocytes (48). 5-LO forms a mo- flammatory conditions are caused by disruptions of this delicate lecular complex with NF-κB and 5-LO inhibitors inhibit NF-κB homeostasis, leading to more active stimulatory pathways and the activation (49). In the MRL/lpr mouse model of SLE, enhanced failure of inflammation resolution. Lipid mediators, such as leu- renal leukotriene production is associated with increased severity kotrienes and SPMs, help maintain immune homeostasis by pro- of lupus nephritis, whereas a leukotriene im- moting inflammation and resolution, respectively. Upsetting their proves renal pathology (50). balance leads to changes in local tissue microenvironments that SPMs are a family of lipids that actively promote resolution alter the timing and duration of inflammation and has been linked and tissue repair without compromising host defenses. Induction to various chronic inflammatory conditions (41). For example, of SPMs has been recognized to have therapeutic potential (51). SLE is a disease of nonresolving inflammation, characterized by SLE patients have lower serum levels of RvD1, suggesting a role of the presence of autoantibodies and systemic inflammation. The SPM dysregulation in the disease (52). We show that RvD1, high percent (80 to 90%) of C1q-deficient individuals develop SLE RvD2, and LXA4 productions are all downstream of HMGB1- clearly shows that C1q helps maintain immune homeostasis (24, plus-C1q stimulation in a RAGE- and LAIR-1–dependent man- 42). Interestingly, there is clinical evidence that derangements in ner. These findings suggest that SPM production is part of the lipid mediators are associated with SLE disease activity. Elevations antiinflammatory polarization of monocytes previously described of urine leukotriene levels are seen in patients with active SLE in HMGB1–C1q cross-linking of RAGE and LAIR-1. It is also (43), and LXA4, an SPM, has been proposed as a biomarker to known that LXA4, RvD1, and RvD2 can activate an M1-to-M2 predict prognosis and response to therapy (44). macrophage phenotype switch in vivo in adipocyte tissue in an We previously described that relative levels of HMGB1 and C1q autocrine or paracrine fashion through engagement of G-protein– reciprocally regulate proinflammatory and proresolving cytokines coupled receptors or formyl peptide receptor 2 (ALX/FPR2) (53). in RAGE- and LAIR-1–dependent pathways (20). Here, we fur- HMGB1-plus-C1q–induced SPM production appears to occur in a ther demonstrate that HMGB1 and C1q utilize the same pathway similar fashion and can contribute to sustaining the antiinflamma- to regulate LTB4 and SPM production through regulation of tory phenotype of monocyte/macrophages. IRF5. These observations both expand on the existing knowledge IRF5 is a member of the IFN regulatory factor family of tran- of lipid mediator pathways and further explore the mechanisms by scription factors that modulate inflammatory immune responses in which HMGB1 and C1q coregulate immune homeostasis, and how numerous cell types (54). It is well established that IRF5 plays an the absence of C1q promotes SLE. important role in various inflammatory conditions. In atheroscle- LTB4 and its activator 5-LO contribute to inflammation through rosis, IRF5 maintains proinflammatory macrophages within ath- increased vascular permeability, attraction, and activation of leu- erosclerotic lesions, impairs efferocytosis, and promotes lesion kocytes (45, 46). It is known that phosphorylation status and cel- growth (55). Pattern recognition receptor (PRR)-stimulated M1 lular localization of 5-LO determine its activity and affect its role macrophages require IRF5 to enhance glycolysis, which is char- in LTB4 versus SPM production (15). Our finding that HMGB1 acteristic of M1 polarization and is a central mediator of inflam- and HMGB1-plus-C1q stimulations result in distinct patterns of mation (56). The role of IRF5 in SLE pathogenesis is well

Fig. 7. Schematic of the IRF5 and LTB4 pathway. HMGB1 engages RAGE and induces both LTB4 production and IRF5 expression. LTB4 binds its receptor to stimulate further induction of IRF5 in a positive-feedback loop, resulting in the additional production of LTB4 and proinflammatory macrophage polarization. HMGB1 plus C1q induces production of specialized proresolving mediators (lipoxin A4, resolvin D1, and resolvin D2), which function to inhibit IRF5 expression through their respective receptors. HMGB1-C1q mimetic (RAGE and LAIR-1 cross-linking peptide [RLCP]) can polarize monocytes to an antiinflammatory phenotype.

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1907490116 Liu et al. Downloaded by guest on September 27, 2021 established. IRF5 leads to production of type I IFN in myeloid cells SLE pathogenesis or other diseases of chronic inflammation. (57). It was shown that IRF5 deficiency ameliorates SLE in mouse Although there are limitations in extrapolating from our short- models (58–60). Moreover, there are IRF5 polymorphisms that term assays to chronic conditions, we showed in our previous study associate with high IRF5 in monocytes and SLE (56, 61). We that these proximal events involving HMGB1 and C1q can have demonstrated a positive-feedback loop between IRF5 and SPMs. impact in adaptive immunity. HMGB1-plus-C1q stimulation sup- LTB4 production depends on IRF5, and that disruption of LTB4 pressed monocyte-to- differentiation and decreased production, by antagonizing or knocking down the LTB4 receptors the ability of macrophages to stimulate T cells (20). Continued BLT1/2, leads to a decrease in IRF5 expression. In contrast, ex- investigation of the immunomodulatory effects of HMGB1 and posure to nanomolar concentrations of SPMs such as RvD1, RvD2, HMGB1 plus C1q can greatly enhance our understanding of SLE and LXA4, decreased HMGB1-stimulated IRF5 expression (Fig. and provide approaches to SLE therapeutics. These findings may 7). These are far more potent than the current drugs used in the also illuminate mechanisms in cancer immunology. It is known that treatment of SLE and other diseases associated with uncontrolled C1q is overexpressed in the stroma and vascular endothelium of inflammation(62).RvD1isanSPMknowntolimit5-LOperi- many malignant tumors and serves as a cancer-promoting factor in nuclear localization and LTB4 synthesis through a calcium-activated tumor microenvironments (69). LAIR-1 is also known to be kinase-dependent pathway (15). Given that IRF5 and LTB4 are overexpressed in several human tumors (70, 71). It is reasonable to codependent and mutually enhancing, it is reasonable to speculate speculate that C1q and LAIR-1 may interact in this context to that the observed decrease in LTB4 following HMGB1-plus-C1q promote tumor escape from immune control, and that the path- stimulation can be attributed, at least in part, to an inhibitory effect ways we have explored are involved in immune function, in general. of SPMs produced by the HMGB1-plus-C1q pathway on 5-LO nuclear localization and phosphorylation, leading to a disrup- Materials and Methods tion of the positive-feedback loop between IRF5 with LTB4. Mice and Study Approval. C57BL/6J WT mice, Lysozyme2-cre mice, and LAIR-1– Although we showed that LTB4 receptor antagonists and deficient C57BL/6 were purchased from The Jackson Laboratory. Lysozyme2-cre resolvins can all decrease HMGB1-induced IRF5 expression, we did mice and LAIR-1–deficient C57BL/6 were bred in our facility to generate mye- not see the same effect in LPS-stimulated monocytes. Thus, the loid cell-specific LAIR-1–deficient mice (LAIR-1 cKO). RAGE-deficient mice and positive-feedback loop between LTB4 and IRF5 does not develop IRF5-deficient mice were maintained in our facility (72, 73). This study was when IRF5 is induced by TLR4 activation (SI Appendix,Fig.S3). Of carried out in strict accordance with recommendations in Guide for the Care and Use of Laboratory Animals of the NIH (74). The protocol was approved by note, the LTB4 receptor, which can associate with RAGE, has not the Institutional Animal Care and Use Committee of the Feinstein Institute for been shown to associate with TLR4 (63). Fully delineating the in- INFLAMMATION

Medical Research. Human study approval: This study is IRB exempt. IMMUNOLOGY AND terrelated pathways involving IRF5 and LTB4 in HMGB1-stimulated monocytes will require further study. Nonetheless, the data presented here provide a mechanism for the coordination of inflammation, with Monocyte Isolation and Cell Culture. Human peripheral blood mononuclear cells were isolated from whole human blood by density gradient centrifugation production of both inflammatory cytokines and inflammatory lipid using Ficoll-Paque Plus (GE Healthcare). Blood was obtained from healthy do- mediators and the coordination of resolution of inflammation, with nors (New York Blood Center). Human approval: This study is IRB exempt. production of both IL-10 and proresolving lipid mediators. Monocytes were enriched by negative selection using a human monocyte en- The RAGE and LAIR-1 cross-linking peptide that mimics the richment kit (Stem Cell Technology). For monocytes from mice, cells were effect of HMGB1 plus C1q not only further confirmed the enriched by positive selection kit (Stem Cell Technology). The purity of mono- downstream effects of RAGE-LAIR-1 cross-linking identified in cytes (≥90%) was determined by flow cytometer (LSRII; BD Biosciences). Purified our study, it also demonstrated that this pathway can be harnessed monocytes (2–5 × 106 cells per mL) were cultured in U-bottom 96-well plates in for therapeutic potential. We showed that RLCP was able to in- X-Vivo 15 serum-free medium (Lonza) with or without HMGB1 (1 μg/mL), C1q duce proresolving macrophage differentiation, increase SPM pro- (25 μg/mL), and RLCP (500 nM) treatment and harvested at the indicated times. duction, and decrease LTB4 production and IRF5 expression both in vitro and in vivo. RLCP induces SPMs and also blocks IRF5 Lipid Mediator Analysis. Culture supernatant or mouse peritoneal lavage was induction. It is important to note that human monocytes use the collected, and LTB4, RvD1, RvD2, and PGE2 were quantitated by ELISA (Cayman Chemical) following the manufacturer’sinstruction.LXA was quantitated eicosanoid oxidoreductase to rapidly inactivate LXA , 4 4 similarly (Oxford Biomedical Research). RvD1, and RvD2 (64–66). The amount of these SPMs induced in vitro were measured in supernatants; therefore, total cellular Statistics. Statistical analyses were performed using Prism 7 (GraphPad), and production of these lipid mediators may be underestimated. values of P < 0.05 were considered significant. Student’s t test analyzed the The 5-LO inhibitor, zileuton, is approved for treating asthma; one variance of mean values between 2 groups for unpaired observations. Group study suggested that it may be effective in managing SLE (67, 68). differences were tested with 1-way ANOVA followed by Tukey’s correction Our identification of RAGE and HMGB1 as upstream compo- for multiple comparisons. nents of 5-LO nuclear translocation and the evidence that HMGB1 plus C1q induce SPMs that suppress leukotriene pro- ACKNOWLEDGMENTS. We thank Heriberto Borrero, Christopher Colon, duction open up opportunities for the designs of therapeutics that Amanda Chan, and Bruce T. Volpe for discussion and technical assistance. This work was supported by grants from the NIH/National Institute of regulate lipid abnormalities in SLE. Together with its observed Arthritis and Musculoskeletal and Skin Diseases (K01AR065506 [M.S.]), the down-regulation of IRF5 and macrophage polarization, RLCP or NIH/National Institute of Allergy and Infectious Diseases (R01AI135063 [M.S.] a functionally similar molecule may address multiple aspects of and P01AI102852 [B.D.]), and the NIH (R01HL127464 and R35HL145228I [I.T.]).

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